Laboratory tests on biological samples have traditionally been used for diagnosis, screening, disease staging, forensic analysis, pregnancy testing, drug testing, and other reasons. While a few qualitative tests, such as pregnancy tests, have been reduced to simple kits for the patient's home use, a large number of quantitative tests still require elaborate procedures that often necessitate the expertise of trained technicians in a laboratory setting using sophisticated instruments. In recent years, some of these tests have been made available for point of care (POC) analysis by using handheld testing devices that embody sensors and computational electronics for sample analysis.
Point of care tests (POCT) are therefore ‘near patient’ diagnostic tests performed outside the routine analytical laboratory. A POCT carried out near the patient is advantageous as it can potentially provide rapid results which enable medical practitioners to act sooner, save lives, improve patient outcomes and reduce overall costs to the healthcare system.
An example of a typical POC device is based on a lateral flow immunoassay that relies on the use of a capture reagent immobilised on a membrane (such as a nitrocellulose strip) to capture an analyte from a sample, which analyte transits the membrane by a process of lateral flow/capillary action. The capture reagent is typically an antibody and the bound analyte is normally detected by means of a second antibody labelled with a visually detectable substance such as colloidal gold. This approach has been widely used in POCTs, for example self-tests for pregnancy involving the detection of human chorionic gonadotrophin (HCG) in urine. Generally speaking, immunoassay POCTs have been used in applications where high sensitivity is not required, so that labels providing visual end points have been highly successful. Results can be reported visually (e.g. HCG pregnancy test) or using an electronic reader (e.g. Alere DDS2 drugs of abuse test system).
It is now widely appreciated that the reduced turnaround time associated with a POCT is a highly desirable goal where extremely high sensitivity of detection is needed to detect trace quantities of certain analytes or compounds. However, conventional lateral flow methods using colloidal gold as an end point cannot provide the required sensitivity. Unfortunately, the high sensitivity of detection provided by routine laboratory analysers often involves complex instrumentation which is not appropriate for near patient testing. This has led to significant and increasing interest in microfluidic (lab on a chip) methods. To date, a very small proportion of microfluidic based POCT concepts have come to market: most notably, the Abbott i-STAT Cardiac Troponin (an enzyme immunoassay carried out on a few drops of blood for early detection of angina and coronary artery occlusion) and the Alere Triage CTnI (fluorescence immunoassay) tests.
Therefore, it is widely acknowledged that current POCTs lack the analytical performance of standard laboratory tests and this leads to a compromise in the value they provide, or has led to complex and expensive systems to achieve such sensitivity. A key example is the measurement of cardiac troponin as a marker for myocardial infarction, where analytical sensitivity can be a factor limiting rapid and accurate diagnosis, thus necessitating the use of a clinical chemistry laboratory. There is therefore a need for a technology that can deliver rapid, high sensitivity POCT in the rapidly growing area of clinical diagnostics.
Chemiluminescence based on acridinium esters (AE) offers one of the most sensitive endpoints developed for immunoassays and genetic probe assays and has been applied in the routine clinical laboratory for a wide range of analytes. The high sensitivity of assays based on AE labels derives from the fact that the chemiluminescence output can be detected against a background that is virtually zero. To take advantage of this sensitivity in practice, there is a requirement that all unreacted label must be removed from the site of reaction prior to initiating the chemiluminescent reaction i.e. the use of AE technology requires a clean separation of signal/background. Thus, conventional assay methods utilising AE labelling techniques typically involve several washing steps to remove unbound AE label (background) and so achieve the desired signal specificity. In this regard typical POCT assay formats do not permit adequate washing of specifically bound labelled antibody to ensure that this requirement for AE detection is met. As a consequence, to date, there have been no successful applications of this sensitive technology in a point-of-care format.
Using novel technology we herein provide a POCT based on a clear separation between signal/background that thus provides the requisite sensitivity for use in a high sensitivity assay.